Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury

Abstract

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1^-/- mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

Fig. 1.. DPEP1 expression in the kidney.

(A) DPEP1 immunohistochemistry in normal human kidney and allograft biopsies with clinical ATN/acute tubular necrosis (ATN). Arrows denote peritubular capillaries. Pt1 and Pt2 denote biopsies from 2 individual patients. Scale bars, 50 μm. Right panels are magnification of hatched boxes. (B and C) Immunoblot probing for DPEP1 in kidney lysates from mice treated with renal IRI and lipopolysaccharide (LPS) at 8 hours. Sham and untreated (NT) mice are controls. (D) DPEP1 immunohistochemistry in wild-type (WT) mouse kidneys following IRI or LPS treatment at 8 hours. Kidney from untreated Dpep1^−/− mouse is used as a control. Arrows denote peritubular capillaries. Scale bars, 50 μm. (E) Kidney IVM in mice following sham operation, LPS administration, and IRI (8 hours). Tubules are visualized using autofluorescence and DPEP1 expression by fluorescent-labeled LSALT peptide. Arrows denote peritubular capillaries. Labels: tubules (green) and DPEP1 (red). Scale bars, 100 μm. Bottom panels are magnification of hatched boxes. (F) Representative flow cytometry for DPEP1 expression in cells isolated from the kidneys of untreated (NT), LPS-, or IRI-treated mice (8 hours). Cells sorted using LTL (proximal tubular epithelial cells), CD31 (endothelial cells), and CD45 (leukocytes).

Fig. 2.. DPEP1 and leukocyte recruitment during kidney IRI.

(A) Kidney IVM in Dpep1^+/+ and Dpep1^−/− mice at 2 hours following renal IRI. Labels: leukocytes (CD11b, red), tubules (autofluorescence, green), and capillaries (QTracker, blue). Scale bars, 100 μm. (B) Quantification of stationary CD11b⁺ cells/field in the kidney (Dpep1^+/+ versus Dpep^1−/−, **P = 0.003, n = 5 to 6 per group, Student’s t test). (C) Kidney IVM in LysM^gfp/gfp mice 2 hours following IRI. Mice were pretreated with vehicle (phosphate-buffered saline), scrambled (Scr) LSALT peptide, or LSALT peptide. Labels: leukocytes (LysM-GFP, bright green) and capillaries (QTracker, blue). Scale bars, 50 μm. (D) Quantification of stationary GFP⁺ leukocytes/field in the kidneys of mice 2 hours after IRI treatment with vehicle, LSALT peptide, or scrambled LSALT peptide (LSALT peptide versus scrambled peptide, *P = 0.03, n = 3 to 5). (E) Kidney IVM and (F) quantification of stationary GFP⁺ leukocytes in LysM^gfp/gfp mice 2 hours after IRI treatment with increasing doses of LSALT peptide. Labels: macrophages (F4/80, red), leukocytes (LysM-GFP, yellow), tubules (autofluorescence, dark green), and capillaries (QTracker, blue) [LSALT peptide 0 μg/kg versus 0.1 μg/kg, ***P = 0.0002; 1 μg/kg, ***P = 0.0001; 10 μg/kg, ***P = 0.0003; 100 μg/kg, ***P = 0.0001; n = 3 to 5 per group, analysis of variance (ANOVA) with Dunnett’s post hoc test].

Fig. 3.. DPEP1-mediated neutrophil and inflammatory monocyte recruitment to the injured kidney.

Flow cytometry of neutrophil (A) and inflammatory monocyte (B) recruitment to the kidneys of Dpep1^+/+ and Dpep1^−/− at 24 hours (circles) and 48 hours (squares) after IRI. Contralateral (Cntrl) kidneys are used as controls. [Neutrophils: Dpep1^+/+ (24 hours) Cntrl versus IRI, **P = 0.005; IRI (24 hours) Dpep1^+/+ versus Dpep1^−/−, **P = 0.008; Dpep1^+/+ (48 hours) Cntrl versus IRI, **P = 0.006; IRI (48 hours) Dpep1^+/+ versus Dpep1^−/−, ***P = 0.001; n = 5 per group, Student’s t test.] [Monocytes: Dpep1^+/+ (24 hours) Cntrl versus IRI, ***P = 0.001; IRI (24 hours) Dpep1^+/+ versus Dpep1^−/−, **P = 0.004; Dpep1^+/+ (48 hours) Cntrl versus IRI, **P = 0.004; IRI (48 hours) Dpep1^+/+ versus Dpep1^−/−, ns = not significant; n = 5 per group, Student’s t test.] Flow cytometry of neutrophil (C) and inflammatory monocyte (D) recruitment to the kidneys of LSALT peptide–treated mice at 24 hours (circles) and 48 hours (squares) after IRI. Contralateral (Cntrl) kidneys are used as controls. [Neutrophils: Cntrl versus IRI (24 hours), **P = 0.005; IRI (24 hours) no treatment versus LSALT, **P = 0.01; Cntrl versus IRI (48 hours), *P = 0.048; IRI (48 hours) no treatment versus LSALT, *P = 0.04; n = 6 to 8 per group, Student’s t test.] [Monocytes: Cntrl versus IRI (24 hours), ***P = 0.0003; IRI (24 hours) no treatment versus LSALT, **P = 0.005; Cntrl versus IRI (48 hours), **P = 0.002; IRI (48 hours) no treatment versus LSALT, *P = 0.047; n = 5 to 8 per group, Student’s t test.]

Fig. 4.. DPEP1 inhibitors and leukocyte recruitment during kidney IRI.

(A) Kidney IVM in LysM^gfp/gfp mice at 2 hours following IRI in mice treated with LSALT peptide, cilastatin, or GFE-1 peptide. Sham-operated kidney is used as a negative control. Scale bars, 100 μm. (B) Stationary GFP⁺ leukocytes/field in the kidney were quantified (versus IRI: cilastatin, ***P = 0.0002; LSALT, ***P = 0.0005; GFE-1, ***P = 0.0003; n = 3 to 5 per group, ANOVA with Dunnett’s post hoc test). (C and D) Flow cytometry of leukocytes isolated from ischemic (IRI) and contralateral (Cntrl) kidneys in wild-type mice treated with or without cilastatin or LSALT peptide at 48 hours (neutrophils: Cntrl versus IRI, ***P = 0.0006; IRI versus IRI + cilastatin, *P = 0.011; IRI versus IRI + LSALT, P = 0.001; n = 5 to 6 per group, ANOVA with Dunnett’s post hoc test) (inflammatory monocytes: Cntrl versus IRI, **P = 0.01; IRI versus IRI + cilastatin, ns = not significant; IRI versus IRI + LSAL, P = 0.047; n = 5 to 6 per group, ANOVA with Dunnett’s post hoc test).

Fig. 5.. Impact of DPEP1 on AKI phenotype during IRI.

(A) Immunofluorescence confocal microscopy for KIM-1 and Ly6G/C in the kidneys of Dpep1^+/+ and Dpep1^−/− mice 48 hours following renal IRI. Labels: KIM-1 (top, red), Ly6G/C (bottom, red), tubules (LTL, green), and nuclei [4′,6-diamidino-2-phenylindole (DAPI), blue]. Scale bars, 50 μm. (B) Renal function determined by serum creatinine in sham- and renal IRI–treated Dpep1^+/+ and Dpep1^−/− mice at 48 hours (IRI Dpep1^+/+ versus Dpep1^−/−, **P = 0.007; n = 8 per group, Student’s t test). (C) Immunofluorescence confocal microscopy for KIM-1 and Ly6G/C in the kidneys of wild-type mice 48 hours following renal IRI with or without treatment with cilastatin or LSALT. Labels: KIM-1 (top, red), Ly6G/C (bottom, red), tubules (LTL, green), and nuclei (DAPI, blue). Scale bars, 50 μm. (D) Renal function determined by serum creatinine in renal IRI–treated mice with or without cilastatin or LSALT peptide at 48 hours (versus IRI alone: cilastatin, **P = 0.005; LSALT, ***P = 0.0005; n = 5 to 14 per group, ANOVA with Dunnett’s post hoc test).

Fig. 6.. Role of CD44 and ICAM-1 in IRI-induced renal inflammation.

(A) IVM in LysM^gfp/gfp mice at 2 hours following renal IRI with and without treatment with anti–ICAM-1, anti-CD44, or LSALT peptide. Labels: leukocytes (LysM-GFP, green), tubules (autofluorescence, dark green), and capillaries (QTracker, blue). Scale bars, 100 μm. (B) Stationary GFP⁺ leukocytes/field in the kidney were quantified (IRI versus anti-CD44: ***P = 0.0002; LSALT: ***P < 0.0001, five per group, ANOVA with Tukey post hoc test). (C) Representative flow cytometry of kidney leukocytes isolated from LysM^gfp/gfp mice 24 hours after IRI with or without LSALT peptide, CD44, or ICAM-1–blocking antibody treatment. Distinct GFP⁺ leukocyte populations were characterized by flow cytometry (GFP^lo = monocytes, GFP^int = macrophages, and GFP^hi = neutrophils). Contralateral kidneys from untreated IRI mice are used as controls. (D) Quantification of kidney GFP⁺ leukocytes in LysM^gfp/gfp mice 24 hours after IRI with and without treatment with LSALT peptide, anti–ICAM-1, and anti-CD44 antibodies. (GFP^int macrophages IRI versus LSALT, ***P = 0.0002; anti–ICAM-1, *P = 0.017; anti–ICAM-1 + LSALT, ***P = 0.0006; anti-CD44, *P = 0.015; anti-CD44 + LSALT, ***P < 0.0001. GFP^hi neutrophils IRI versus LSALT, *P = 0.037; anti–ICAM-1, P = ns; anti–ICAM-1 + LSALT, *P = 0.034; anti-CD44, **P = 0.001; anti-CD44 + LSALT, ***P < 0.0001. Four to six per group, ANOVA with Dunnett’s post hoc test.)

Fig. 7.. DPEP1-mediated leukocyte recruitment and tubular injury.

(A) Immunoblotting for LRP2, GPX4, ACSL4, and DPEP1 expression in whole-kidney tissue from Dpep1^−/− and Dpep1^+/+ mice 48 hours after renal IRI or sham operation. (B) Kidney IVM in LysM^gfp/gfp mice at 90 min following LPS administration with or without cilastatin or LSALT peptide treatment. Scale bars, 100 μm. (C) Stationary GFP⁺ leukocytes/field in the kidney were quantified (versus LPS: cilastatin, *P = 0.01; LSALT, **P = 0.002; n = 4 to 5 per group, ANOVA with Dunnett’s post hoc test). (D) Kidney IVM in Dpep1^+/+ and Dpep1^−/− mice at 90 min following LPS administration. Labels: leukocytes (CD11b, red), capillaries (QTracker, blue), and tubules (autofluorescence, yellow-green). Scale bars, 100 μm. (E) Kidney IVM with SYTOX Red staining in LysM^gfp/gfp mice 2 hours following LPS administration. Non-LPS (NT)–treated mice are shown as a control. Labels: leukocytes (LysM-GFP, bright green/yellow), tubules (autofluorescence, dark green), capillaries (QTracker, blue), and necrotic cells (SYTOX, red). Scale bars, 100 μm.